PROJECT SUMMARY Notch signaling is critical for early T-cell development. At its core, Notch signaling relies on the interaction between Notch-family receptors with their cognate, transmembrane-bound Delta-like family ligands. Upon ligand recognition and binding, Notch receptors undergo a series of proteolytic cleavage events, allowing the release and translocation of Notch intracellular domain (NotchICD) into the cell nucleus. Within the nucleus, NotchICD can interact with its DNA-binding partner, Recombination Signal Binding Protein-Jk (RBPJk) to activate expression of target genes. Despite the essential role for Notch-mediated gene regulation in this developmental context, it remains a mystery how NotchICD can localize to DNA and differentially regulate gene expression, given its near-uniform expression pattern. One important negative regulator of Notch signaling is Spen. Spen has been shown to compete with NotchICD for RBPJk binding and can recruit co-repressive complexes to remodel chromatin and repress genomic loci. Recently, Spen was shown to directly interact with a group of nucleic acid molecules called long non-coding RNA (lncRNA). Given their preferential localization within the cell nucleus, ability to coordinate nuclear organization through protein-scaffolding, and documented tissue and temporal specificity greater than mRNA or protein, lncRNAs are an attractive and as of yet- uncharacterized class of regulatory molecules that could mediate differential expression of Notch-target genes. We hypothesize that lncRNAs recruit Spen to distinct RBPJk bound sites on DNA. This in turn, biases NotchICD towards sites of `un-occupied' RBPJk, leading to activation of target genes. Changes in lncRNA expression would modulate Spen localization and alter NotchICD's accessible genomic repertoire in a stage- and temporally-specific manner. We will this hypothesis by characterizing the dynamics of NotchICD and Spen genomic localization using Chromatin-ImmunoPrecipitation, determining if Spen's interactions with RNA are critical for regulation of Notch signaling using Spen truncation analysis, and characterizing the roles of specific lncRNA-Spen interactions on differential expression of Notch target genes using genetic perturbation, in ex vivo differentiated pro-T cell subsets. The results of this study have the potential to incorporate lncRNAs into our understanding of dynamic gene regulation during development and differentiation, generalizable to signaling pathways in other biologic contexts. Moreover, dysregulation of the Notch signaling pathway has been linked to a variety of hematologic and solid malignancies, including T-Acute Lymphoblastic Leukemia and Pancreatic cancer. By identifying and characterizing novel lncRNA-Spen interactions in this study, we hope to identify new, increasingly specific targets for pharmacologic manipulation of the Notch signaling pathway towards the treatment of human disease.